Nucleic acid sequences and methods of use for the production of plants with modified polyunsaturated fatty acid levels

ABSTRACT

By this invention, novel nucleic acid sequences are provided, wherein said nucleic acid sequence is a genomic sequence of a plant desaturase encoding sequence. Also provided in the present invention are the promoter and intron sequences of the desaturase genomic sequences. Furthermore, recombinant DNA constructs employing the polynucleotide sequences are provided. The instant invention also provides methods for the modification of fatty acid compositions in host plant cells.

INTRODUCTION

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/151,224 filed Aug. 26, 1999 and U.S. ProvisionalApplication Serial No. 60/172,128 filed Dec. 17, 1999.

TECHNICAL FIELD

[0002] The present invention is directed to nucleic acid sequences andconstructs, and methods related thereto.

BACKGROUND

[0003] Plant oils are used in a variety of applications. Novel vegetableoils compositions and/or improved means to obtain oils compositions,from biosynthetic or natural plant sources, are needed. Depending uponthe intended oil use, various different fatty acid compositions aredesired.

[0004] One means postulated to obtain such oils and/or modified fattyacid compositions is through the genetic engineering of plants. However,it is necessary to identify the appropriate nucleic acid sequences whichare capable of producing the desired phenotypic result, regulatoryregions capable of directing the correct application of such sequences,and the like.

[0005] Higher plants appear to synthesize fatty acids via a commonmetabolic pathway (fatty acid synthetase pathway). In developing seeds,where fatty acids are attached to glycerol backbones, formingtriglycerides, for storage as a source of energy for furthergermination, the FAS pathway is located in the proplastids. The firstcommitted step is the formation of acetyl-ACP (acyl carrier protein)from acetyl-CoA and ACP catalyzed by the enzyme, acetyl-CoA:ACPtransacylase (ATA). Elongation of acetyl-ACP to 16- and 18-carbon fattyacids involves the cyclical action of the following sequence ofreactions: condensation with a two-carbon unit from malonyl-ACP to forma β-ketoacyl-ACP (β-ketoacyl-ACP synthase), reduction of theketo-function to an alcohol (β-ketoacyl-ACP reductase), dehydration toform an enoyl-ACP (β-hydroxyacyl-ACP dehydrase), and finally reductionof the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACPreductase). β-ketoacyl-ACP synthase I, catalyzes elongation up topalmitoyl-ACP (C16:0), whereas β-ketoacyl-ACP synthase II catalyzes thefinal elongation to stearoyl-ACP (C18:0). Common plant unsaturated fattyacids, such as oleic, linoleic and a-linolenic acids found in storagetriglycerides, originate from the desaturation of stearoyl-ACP to formoleoyl-ACP (C18:1) in a reaction catalyzed by a soluble plastid Δ-9desaturase (also often referred to as “stearoyl-ACP desaturase”).Molecular oxygen is required for desaturation in which reducedferredoxin serves as an electron co-donor. Additional desaturation iseffected sequentially by the actions of membrane bound Δ-12 desaturaseand Δ-15 desaturase. These “desaturases” thus create mono- orpolyunsaturated fatty acids respectively.

[0006] Obtaining nucleic acid sequences capable of producing aphenotypic result in FAS, desaturation and/or incorporation of fattyacids into a glycerol backbone to produce an oil is subject to variousobstacles including but not limited to the identification of metabolicfactors of interest, choice and characterization of an enzyme sourcewith useful kinetic properties, purification of the protein of interestto a level which will allow for its amino acid sequencing, utilizingamino acid sequence data to obtain a nucleic acid sequence capable ofuse as a probe to retrieve the desired DNA sequence, and the preparationof constructs, transformation and analysis of the resulting plants.

[0007] Thus, additional nucleic acid targets and methods for modifyingfatty acid compositions are needed. In particular, constructs andmethods to produce a variety of ranges of different fatty acidcompositions are needed.

SUMMARY OF THE INVENTION

[0008] The present invention is generally directed to genomic desaturasepolynucleotides, and in particular to genomic desaturase polynucleotideswhich encode enzymes that catalyze the insertion of a double bond into afatty acyl moiety at the twelfth (Δ12 desaturase or fad2) or fifteenth(Δ15 desaturase or fad3) carbon position in a fatty acyl chain ascounted from the carboxyl terminus. Further, the present inventionprovides isolated non-coding regions of such genomic polynucleotidesequences, particularly including the introns, and promoter regions.Specific oligonucleotides are provided which include partial or completesequences which are derived from Δ12 and Δ15 desaturase promoter andintron sequences. Although the sequences disclosed herein are obtainedfrom soybean plants, it is contemplated that additional sequences can bederived from intron and promoter regions of desaturase genomicpolynucleotide sequences which are homologous or have identity to thesoybean desaturase sequences. Such additional desaturase sequences canbe obtained using standard methods described below from a variety ofplant sources, in particular oilseed crops.

[0009] It is also an aspect of the present invention to providerecombinant DNA constructs which can be used for the modification of thefatty acid composition in a plant and in particular, to modify thetranscription or transcription and translation (expression) ofdesaturase genes or proteins, such as Δ12 and Δ15 desaturase. Theinvention is particularly directed to DNA constructs which includesequences which are derived from the intron or promoter regions of agenomic clone wherein said sequences are in a sense or antisenseorientation in a DNA construct. These DNA constructs are then used totransform or transfect host cells to produce plants with modified levelsof fatty acids, particularly modified levels of oleic, linoleic andlinolenic acid. It is particularly contemplated to provide constructsand methods for down regulating Δ12 and Δ15 desaturase gene expression,so as to increase the levels of oleic acid and to decrease the levels oflinoleic acid and linolenic acid. It is particularly contemplated toalter the fatty acid composition in seed tissue of oilseed crops.

[0010] The modified plant cells, plants, seeds and oils obtained by theexpression of the Δ12 and Δ15 desaturase polynucleotides are alsoconsidered part of the invention. Further, it is contemplated to produceoil compositions with specific relative levels of each fatty acid. Onepreferred embodiment comprises at least about 80-85% oleic acid, no morethan about 1-2% linoleic acid, and no more than about 1-3% linolenicacid; and a second preferred embodiment comprising at least about 50-75%oleic acid, at least about 10-30% linoleic acid, and no more than about3% linolenic acid.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention is directed to genomic desaturasesequences, particularly the isolated non-coding sequences from genomicfatty acid desaturase nucleic acid sequences from host cell sources. Adesaturase sequence of this invention includes any nucleic acid genomicsequence, including all non-coding regions, encoding amino acids from asource, such as a protein, polypeptide or peptide, obtainable from acell source, which is capable of catalyzing the insertion of a doublebond into a fatty acyl moiety in a plant host cell, i.e., in vivo, or ina plant cell-like environment, i.e. in vitro. As will be described inmore detail below, specific genomic polynucleotide sequences encodingenzymes which add double bonds at the twelfth (Δ12 desaturase) andfifteenth (Δ15 desaturase) carbon positions in a fatty acyl chain ascounted from the carboxyl terminus are provided. In addition, providedherein are specific non-coding regions of such genomic sequences.

[0012] The term “non-coding” refers to sequences of polynucleotides thatdo not encode part or all of an expressed protein. Non-coding sequencesinclude but are not limited to introns, promoter regions, and 5′untranslated regions.

[0013] The term “intron” as used herein refers to the normal sense ofthe term as meaning a segment of polynucleotides, usually DNA, that doesnot encode part or all of an expressed protein.

[0014] The term “exon” as used herein refers to the normal sense of theterm as meaning a segment of polynucleotides, usually DNA, that encodespart or all of an expressed protein.

[0015] Thus, the term “intron” refers to gene regions that aretranscribed into RNA molecules, but which are spliced out of the RNAbefore the RNA is translated into a protein. As contrasted to the term“exon” which refers to gene regions that are transcribed into RNA andsubsequently translated into proteins.

[0016] As set forth in detail in the sequence listing and the examples,genomic Δ12 desaturase and Δ15 desaturase sequences and intron andpromoter regions obtained from such sequences are provided herein. Inparticular, two Δ12 desaturase genomic clones were identified and areset forth in SEQ ID NOs:1 and 23. A single Δ15 desaturase genomic clonewas identified and is set forth in SEQ ID NO:3. A single intron regionwas obtained from each of the Δ12 desaturase genomic clones with thesequences provided in SEQ ID NOs:2 and 24, respectively. The promoterregion from each of the Δ12 desaturase genomic clones are respectivelyincluded in SEQ ID NO:1 (base pairs 1-1094) and SEQ ID NO:23 (base pairs1-1704). The Δ15 desaturase included seven introns in the coding region(set forth as SEQ ID NOs:4, 5, 6, 7, 8, 25 and 26). In addition,preliminary results suggest that there is an additional intron withinthe 5′ untranslated region.

[0017] Although the sequences described herein are obtained fromsoybean, it is contemplated that intron and promoter regions can beobtained from desaturase genomic polynucleotide sequences which arehomologous or have identity to the soybean desaturase sequences. Inparticular, sequences can be obtained from other plant sources andparticularly from oilseed crops. Such genomic sequences can be obtainedusing standard methods, certain of which are described below.

[0018] The sequences of the present invention can be used to modify thefatty acid composition in a plant (see Example 3 and Table I). Inparticular, it is shown that sense and antisense suppression can be usedto obtain broad ranges in the levels of oleic, linoleic and linolenicacid. In particular, it is shown that levels of oleic acid can rangefrom about 26 to 80%, levels of linoleic acid can range from about 2.97to 49.92% and levels of linolenic acid can range from about 3.38 to8.81%. However, these are merely representative of the broad range thatbe can achieved. Moreover, it is contemplated that combinations of thesequences could be used to achieve additional fatty acid compositions.Certain compositions are preferred based on the intended use of the oil.

[0019] One preferred composition includes at least about 50-75% oleicacid, at least about 10-30% linoleic acid and no more than about 3%linolenic acid. A particularly preferred embodiment includes at leastabout 60-70% oleic acid, at least about 15-20% linoleic acid and no morethan about 3% linolenic acid.

[0020] Although the examples set forth herein utilize sense or antisensesuppression to downregulate the gene of interest, it is contemplatedthat other means of modifying gene expression can be used. Inparticular, it is contemplated that gene expression can be downregulated using DNA binding proteins which can be designed tospecifically bind to the non-coding regions identified herein or thatribozymes can be designed to cleave such non-coding regions. Inaddition, as described below, other methods of downregulation of geneexpression which are well known in the art are contemplated and can beused with the sequences of the present invention.

[0021] Isolated Polynucleotides, Proteins, and Polypeptides

[0022] A first aspect of the present invention relates to isolateddesaturase polynucleotides. The polynucleotide sequences of the presentinvention include isolated polynucleotides that are obtainable fromgenomic nucleic acid sequences.

[0023] The invention provides a polynucleotide sequence identical overits entire length to each sequence as set forth in the Sequence Listing.The polynucleotide includes non-coding sequences, including for example,but not limited to, non-coding 5′ and 3′ sequences, such as thetranscribed, untranslated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence that encodes additional aminoacids. For example, a marker sequence can be included to facilitate thepurification of the fused polypeptide. Polynucleotides of the presentinvention also include polynucleotides comprising a structural gene andthe naturally associated sequences that control gene expression.

[0024] The invention also includes polynucleotides of the formula:

X−(R₁)_(n)−(R₂)−(R₃)_(n)−Y

[0025] wherein, at the 5′ end, X is hydrogen, and at the 3′ end, Y ishydrogen or a metal, R₁ and R₃ are any nucleic acid residue, n is aninteger between 1 and 3000, preferably between 1 and 1000 and R₂ is anucleic acid sequence of the invention, particularly a nucleic acidsequence selected from the group set forth in the Sequence Listing andpreferably SEQ ID NOs: 1-8, and 23-29. In the formula, R₂ is oriented sothat its 5′ end residue is at the left, bound to R₁, and its 3′ endresidue is at the right, bound to R₃. Any stretch of nucleic acidresidues denoted by either R group, where R is greater than 1, may beeither a heteropolymer or a homopolymer, preferably a heteropolymer.

[0026] Further preferred embodiments of the invention that are at least50%, 60%, or 70% identical over their entire length to a polynucleotideof the invention, and polynucleotides that are complementary to suchpolynucleotides. More preferable are polynucleotides that comprise aregion that is at least 80% identical over its entire length to apolynucleotide of the invention and polynucleotides that arecomplementary thereto. In this regard, polynucleotides at least 90%identical over their entire length are particularly preferred, those atleast 95% identical are especially preferred. Further, those with atleast 97% identity are highly preferred and those with at least 98% and99% identity are particularly highly preferred, with those at least 99%being the most highly preferred.

[0027] Preferred embodiments are polynucleotides that are obtained fromgenomic polynucleotide sequences and set forth in the Sequence Listing.

[0028] The invention further relates to polynucleotides that hybridizeto the above-described sequences. In particular, the invention relatesto polynucleotides that hybridize under stringent conditions to theabove-described polynucleotides. As used herein, the terms “stringentconditions” and “stringent hybridization conditions” mean thathybridization will generally occur if there is at least 95% andpreferably at least 97% identity between the sequences. An example ofstringent hybridization conditions is overnight incubation at 42° C. ina solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmonsperm DNA, followed by washing the hybridization support in 0.1×SSC atapproximately 65° C. Other hybridization and wash conditions are wellknown and are exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, cold Spring Harbor, N.Y. (1989),particularly Chapter 11.

[0029] The invention also provides a polynucleotide consistingessentially of a polynucleotide sequence obtainable by screening anappropriate library containing the complete gene for a polynucleotidesequence set forth in the Sequence Listing under stringent hybridizationconditions with a probe having the sequence of said polynucleotidesequence or a fragment thereof; and isolating said polynucleotidesequence. Fragments useful for obtaining such a polynucleotide include,for example, probes and primers as described herein.

[0030] As discussed herein regarding polynucleotide assays of theinvention, for example, polynucleotides of the invention can be used asa hybridization probe for RNA, cDNA, or genomic DNA to isolate fulllength cDNAs or genomic clones encoding a polypeptide and to isolatecDNA or genomic clones of other genes that have a high sequencesimilarity to a polynucleotide set forth in the Sequence Listing. Suchprobes will generally comprise at least 15 bases. Preferably such probeswill have at least 30 bases and can have at least 50 bases. Particularlypreferred probes will have between 30 bases and 50 bases, inclusive.

[0031] The region of each gene that comprises or is comprised by apolynucleotide sequence set forth in the Sequence Listing may beisolated by screening using a DNA sequence provided in the SequenceListing to synthesize an oligonucleotide probe. A labeledoligonucleotide having a sequence complementary to that of apolynucleotide of the invention is then used to screen a library ofcDNA, genomic DNA or mRNA to identify members of the library whichhybridize to the probe. For example, synthetic oligonucleotides areprepared which correspond to the desaturase promoter and intronsequences. In particular, screening of cDNA libraries in phage vectorsis useful in such methods due to lower levels of backgroundhybridization.

[0032] Typically, a desaturase sequence obtainable from the use ofnucleic acid probes will show 60-70% sequence identity between thetarget desaturase sequence and the encoding sequence used as a probe.However, lengthy sequences with as little as 50-60% sequence identitymay also be obtained. The nucleic acid probes may be a lengthy fragmentof the nucleic acid sequence, or may also be a shorter, oligonucleotideprobe. When longer nucleic acid fragments are employed as probes(greater than about 100 bp), one may screen at lower stringencies inorder to obtain sequences from the target sample which have 20-50%deviation (i.e., 50-80% sequence homology) from the sequences used asprobe. Oligonucleotide probes can be considerably shorter than theentire nucleic acid sequence encoding an desaturase enzyme, but shouldbe at least about 10, preferably at least about 15, and more preferablyat least about 20 nucleotides. A higher degree of sequence identity isdesired when shorter regions are used as opposed to longer regions. Itmay thus be desirable to identify regions of highly conserved amino acidsequence to design oligonucleotide probes for detecting and recoveringother related desaturase genes. Shorter probes are often particularlyuseful for polymerase chain reactions (PCR), especially when highlyconserved sequences can be identified. (See, Gould, et al., PNAS USA(1989) 86:1934-1938.).

[0033] “Identity”, as is well understood in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as determined by the matchbetween strings of such sequences. “Identity” can be readily calculatedby known methods including, but not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York (1988); Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M. and Griffin, H. G., eds., HumanaPress, New Jersey (1994); Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., Stockton Press, New York (1991); and Carillo,H., and Lipman, D., SIAM J Applied Math, 48:1073 (1988). Methods todetermine identity are designed to give the largest match between thesequences tested. Moreover, methods to determine identity are codifiedin publicly available programs. Computer programs which can be used todetermine identity between two sequences include, but are not limitedto, GCG (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984);suite of five BLAST programs, three designed for nucleotide sequencesqueries (BLASTN, BLASTX, and TBLASTX) and two designed for proteinsequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology,12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)).The BLAST X program is publicly available from NCBI and other sources(BLAST Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol., 215:403-410 (1990)). The well knownSmith Waterman algorithm can also be used to determine identity.

[0034] Parameters for polypeptide sequence comparison typically includethe following:

[0035] Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

[0036] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci USA 89:10915-10919 (1992)

[0037] Gap Penalty: 12

[0038] Gap Length Penalty: 4

[0039] A program which can be used with these parameters is publiclyavailable as the “gap” program from Genetics Computer Group, MadisonWis. The above parameters along with no penalty for end gap are thedefault parameters for peptide comparisons.

[0040] Parameters for polynucleotide sequence comparison include thefollowing:

[0041] Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

[0042] Comparison matrix: matches=+10; mismatches=0

[0043] Gap Penalty: 50

[0044] Gap Length Penalty: 3

[0045] A program which can be used with these parameters is publiclyavailable as the “gap” program from Genetics Computer Group, MadisonWisconsin. The above parameters are the default parameters for nucleicacid comparisons.

[0046] For immunological screening, antibodies to the protein can beprepared by injecting rabbits or mice with the purified protein orportion thereof, such methods of preparing antibodies being well knownto those in the art. Either monoclonal or polyclonal antibodies can beproduced, although typically polyclonal antibodies are more useful forgene isolation. Western analysis may be conducted to determine that arelated protein is present in a crude extract of the desired plantspecies, as determined by cross-reaction with the antibodies to theencoded proteins. When cross-reactivity is observed, genes encoding therelated proteins are isolated by screening expression librariesrepresenting the desired plant species. Expression libraries can beconstructed in a variety of commercially available vectors, includinglambda gt11, as described in Sambrook, et al. (Molecular Cloning: ALaboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

[0047] Plant Constructs and Methods of Use

[0048] Of particular interest is the use of the polynucleotide sequencesin recombinant DNA constructs to direct the transcription of thedesaturase genomic sequences of the present invention in a host plantcell. The expression constructs generally comprise a promoter functionalin a host plant cell operably linked to a nucleic acid sequence of thepresent invention and a transcriptional termination region functional ina host plant cell.

[0049] Those skilled in the art will recognize that there are a numberof promoters which are functional in plant cells, and have beendescribed in the literature. Chloroplast and plastid specific promoters,chloroplast or plastid functional promoters, and chloroplast or plastidoperable promoters are also envisioned.

[0050] One set of promoters are constitutive promoters such as theCaMV35S or FMV35S promoters that yield high levels of expression in mostplant organs. Enhanced or duplicated versions of the CaMV35S and FMV35Spromoters are useful in the practice of this invention (Odell, et al.(1985) Nature 313:810-812; Rogers, U.S. Pat. No. 5,378,619). Inaddition, it may also be preferred to bring about expression of thesequences of the present invention in specific tissues of the plant,such as leaf, stem, root, tuber, seed, fruit, etc., and the promoterchosen should have the desired tissue and developmental specificity.

[0051] Of particular interest is the expression of the nucleic acidsequences of the present invention from transcription initiation regionswhich are preferentially expressed in a plant seed tissue. Examples ofsuch seed preferential transcription initiation sequences include thosesequences derived from sequences encoding plant storage protein genes orfrom genes involved in fatty acid biosynthesis in oilseeds. Examples ofsuch promoters include the 5′ regulatory regions from such genes asnapin (Kridl et al., Seed Sci. Res. 1:209:219 (1991)), phaseolin, zein,soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, soybean α′subunit of β-conglycinin (soy 7s, (Chen et al, Proc. Natl. Acad. Sci.,83:8560-8564 (1986))) and oleosin.

[0052] It may be advantageous to direct the localization of proteinsconferring desaturase to a particular subcellular compartment, forexample, to the mitochondrion, endoplasmic reticulum, vacuoles,chloroplast or other plastidic compartment. For example, where the genesof interest of the present invention will be targeted to plastids, suchas chloroplasts, for expression, the constructs will also employ the useof sequences to direct the gene to the plastid. Such sequences arereferred to herein as chloroplast transit peptides (CTP) or plastidtransit peptides (PTP). In this manner, where the gene of interest isnot directly inserted into the plastid, the expression construct willadditionally contain a gene encoding a transit peptide to direct thegene of interest to the plastid. The chloroplast transit peptides may bederived from the gene of interest, or may be derived from a heterologoussequence having a CTP. Such transit peptides are known in the art. See,for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126;Clark et al. (1989) J. Biol. Chem. 264:17544-17550; della-Cioppa et al.(1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys.Res Commun. 196:1414-1421; and, Shah et al. (1986) Science 233:478-481.

[0053] Depending upon the intended use, the constructs may contain theentire genomic nucleic acid sequence or a particular non-coding regionof such a sequence or a portion of such sequences. For example, whereantisense inhibition of a given desaturase protein is desired, theentire sequence is not required. Furthermore, where desaturase sequencesused in constructs are intended for use as probes, it may beadvantageous to prepare constructs containing only a particular portionof a desaturase sequence, for example a sequence which encodes a highlyconserved desaturase region.

[0054] The skilled artisan will recognize that there are various methodsfor the inhibition of expression of endogenous sequences in a host cell.Such methods include, but are not limited to, antisense suppression(Smith, et al. (1988) Nature 334:724-726), co-suppression (Napoli, etal. (1989) Plant Cell 2:279-289), ribozymes (PCT Publication WO97/10328), combinations of sense and antisense (Waterhouse, et al.(1998) Proc. Natl. Acad. Sci. USA 95:13959-13964), promoter silencing(Park, et al. (1996) Plant J. 9(2):183-194), DNA binding proteins(Beerli, et al. (1997) Proc. Natl. Acad. Sci. USA, 95:14628-14633; andLiu, et al. (1998) Proc. Natl. Acad. Sci. USA, 94:5525-5530). Methodsfor the suppression of endogenous sequences in a host cell typicallyemploy the transcription or transcription and translation of at least aportion of the sequence to be suppressed. Such sequences may behomologous to coding as well as non-coding regions of the endogenoussequence.

[0055] Regulatory transcript termination regions may be provided inplant expression constructs of this invention as well. Transcripttermination regions may be provided by the DNA sequence encoding thedesaturase or a convenient transcription termination region derived froma different gene source, for example, the transcript termination regionwhich is naturally associated with the transcript initiation region. Theskilled artisan will recognize that any convenient transcripttermination region which is capable of terminating transcription in aplant cell may be employed in the constructs of the present invention.

[0056] Alternatively, constructs may be prepared to direct theexpression of the desaturase sequences directly from the host plant cellplastid. Such constructs and methods are known in the art and aregenerally described, for example, in Svab, et al. (1990) Proc. Natl.Acad. Sci. USA 87:8526-8530 and Svab and Maliga (1993) Proc. Natl. Acad.Sci. USA 90:913-917 and in U.S. Pat. No. 5,693,507.

[0057] A plant cell, tissue, organ, or plant into which the recombinantDNA constructs containing the expression constructs have been introducedis considered transformed, transfected, or transgenic. A transgenic ortransformed cell or plant also includes progeny of the cell or plant andprogeny produced from a breeding program employing such a transgenicplant as a parent in a cross and exhibiting an altered phenotyperesulting from the presence of a desaturase nucleic acid sequence.

[0058] Plant expression or transcription constructs having a desaturasepolynucleotide of the present invention as the DNA sequence of interestfor increased or decreased expression thereof may be employed with awide variety of plant life, particularly, plant life involved in theproduction of vegetable oils for edible and industrial uses. Mostespecially preferred are temperate oilseed crops. Plants of interestinclude, but are not limited to, rapeseed (Canola and High Erucic Acidvarieties), sunflower, safflower, cotton, soybean, peanut, coconut andoil palms, and corn. Depending on the method for introducing therecombinant constructs into the host cell, other DNA sequences may berequired. Importantly, this invention is applicable to dicotyledons andmonocotyledons species alike and will be readily applicable to newand/or improved transformation and regulation techniques.

[0059] Of particular interest, is the use of plant desaturase promoterand/or intron constructs in plants to produce plants or plant parts,including, but not limited to leaves, stems, roots, reproductive, andseed, with a modified fatty acid composition. Of particular interest inthe desaturase promoter and/or intron constructs is the use of thepromoter and/or intron sequences of the Δ-12 and Δ-15 desaturase genomicsequences in sense or antisense orientations for the modification offatty acid compositions in host cells.

[0060] The polynucleotides of the present invention can be used in thepreparation of constructs for use in a variety of host cells. Host foruse in the present invention include, but are not limited to plantcells, bacterial cells, fungal cells (including yeast), insect cells,and mammalian cells.

[0061] For example, to confirm the activity and specificity of theproteins encoded by the identified nucleic acid sequences as desaturaseenzymes, in vitro assays can be performed in insect cell cultures usingbaculovirus expression systems. Such baculovirus expression systems areknown in the art and are described by Lee, et al. U.S. Pat. No.5,348,886, the entirety of which is herein incorporated by reference.

[0062] The method of transformation in obtaining such transgenic plantsis not critical to the instant invention, and various methods of planttransformation are currently available. Furthermore, as newer methodsbecome available to transform crops, they may also be directly appliedhereunder. For example, many plant species naturally susceptible toAgrobacterium infection may be successfully transformed via tripartiteor binary vector methods of Agrobacterium mediated transformation. Inmany instances, it will be desirable to have the construct bordered onone or both sides by T-DNA, particularly having the left and rightborders, more particularly the right border. This is particularly usefulwhen the construct uses A. tumefaciens or A. rhizogenes as a mode fortransformation, although the T-DNA borders may find use with other modesof transformation. In addition, techniques of microinjection, DNAparticle bombardment, and electroporation have been developed whichallow for the transformation of various monocot and dicot plant species.

[0063] Normally, included with the DNA construct will be a structuralgene having the necessary regulatory regions for expression in a hostand providing for selection of transformant cells. The gene may providefor resistance to a cytotoxic agent, e.g. antibiotic, heavy metal,toxin, etc., complementation providing prototrophy to an auxotrophichost, viral immunity or the like. Depending upon the host species theexpression construct or components thereof are introduced, one or moremarkers may be employed, where different conditions for selection areused for the different hosts.

[0064] Where Agrobacterium is used for plant cell transformation, avector may be used which may be introduced into the Agrobacterium hostfor homologous recombination with T-DNA or the Ti- or Ri-plasmid presentin the Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNAfor recombination may be armed (capable of causing gall formation) ordisarmed (incapable of causing gall formation), the latter beingpermissible, so long as the vir genes are present in the transformedAgrobacterium host. The armed plasmid can give a mixture of normal plantcells and gall.

[0065] In some instances where Agrobacterium is used as the vehicle fortransforming host plant cells, the expression or transcription constructbordered by the T-DNA border region(s) will be inserted into a broadhost range vector capable of replication in E. coli and Agrobacterium,there being broad host range vectors described in the literature.Commonly used is pRK2 or derivatives thereof. See, for example, Ditta,et al., (Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0120 515, which are incorporated herein by reference. Alternatively, onemay insert the sequences to be expressed in plant cells into a vectorcontaining separate replication sequences, one of which stabilizes thevector in E. coli, and the other in Agrobacterium. See, for example,McBride and Summerfelt (Plant Mol. Biol. (1990) 14:269-276), wherein thepRiHRI (Jouanin, et al., Mol. Gen. Genet. (1985) 201:370-374) origin ofreplication is utilized and provides for added stability of the plantexpression vectors in host Agrobacterium cells.

[0066] Included with the expression construct and the T-DNA will be oneor more markers, which allow for selection of transformed Agrobacteriumand transformed plant cells. A number of markers have been developed foruse with plant cells, such as resistance to chloramphenicol, kanamycin,the aminoglycoside G418, hygromycin, or the like. The particular markeremployed is not essential to this invention, one or another marker beingpreferred depending on the particular host and the manner ofconstruction.

[0067] For transformation of plant cells using Agrobacterium, explantsmay be combined and incubated with the transformed Agrobacterium forsufficient time for transformation, the bacteria killed, and the plantcells cultured in an appropriate selective medium. Once callus forms,shoot formation can be encouraged by employing the appropriate planthormones in accordance with known methods and the shoots transferred torooting medium for regeneration of plants. The plants may then be grownto seed and the seed used to establish repetitive generations and forisolation of vegetable oils.

[0068] For the alteration of unsaturated fatty acid production in a hostcell, a second expression construct can be used in accordance with thepresent invention. For example, the desaturase expression construct canbe introduced into a host cell in conjunction with a second expressionconstruct having a nucleotide sequence for a protein involved in fattyacid biosynthesis.

[0069] There are several possible ways to obtain the plant cells of thisinvention which contain multiple expression constructs. Any means forproducing a plant comprising a construct having a DNA sequence encodingthe expression construct of the present invention, and at least oneother construct having another DNA sequence encoding an enzyme areencompassed by the present invention. For example, the expressionconstruct can be used to transform a plant at the same time as thesecond construct either by inclusion of both expression constructs in asingle transformation vector or by using separate vectors, each of whichexpress desired genes. The second construct can be introduced into aplant which has already been transformed with the desaturase expressionconstruct, or alternatively, transformed plants, one expressing thedesaturase construct and one expressing the second construct, can becrossed to bring the constructs together in the same plant.

[0070] The invention now being generally described, it will be morereadily understood by reference to the following examples which areincluded for purposes of illustration only and are not intended to limitthe present invention.

EXAMPLES Example 1 Cloning of Desaturase Genomic Sequences

[0071] 1A. Soybean Δ-12 Desaturase (fad2-1)

[0072] The soybean fad 2-1A sequence was identified by screening asoybean genomic library using a soybean fad2-1 cDNA probe. Threeputative soy fad 2-1 clones were identified and plaque purified. Two ofthe three soy fad 2-1 clones were ligated into pBluescript II KS+(Stratagene) and sequenced. Both clones (14-1 and 11-12) were the sameand matched the soy fad 2-1 cDNA exactly. The sequence of the entirefad2-1A clone is provided in SEQ ID NO: 1.

[0073] Prior to obtaining this full length clone, a portion of thefad2-1A genomic clone was PCR amplified using PCR primers designed fromthe 5′ untranslated sequence (Primer 12506, 5′-ATACAA GCCACTAGGCAT-3′,SEQ ID NO:9) and within the cDNA (Primer 11698:5′-GATTGGCCATGCAATGAGGGAAAAGG-3′, SEQ ID NO:10. The resulting PCRproduct, which contained the fad2-1A intron, was cloned into the vectorpCR 2.1 (Invitrogen) and sequenced. The soy fad 2-1A partial genomicclone(SEQ ID NO:27) and its intron region (SEQ ID NO:2) were identifiedby comparison to the soybean cDNA sequence using the Pustell comparisonprogram in Macvector. The intron sequence begins after the ATG startcodon, and is 420 bases long.

[0074] A second fad2-1 gene family member was also identified andcloned, and is referred to herein as fad2-1B. The soy fad 2-1B partialgenomic clone (SEQ ID NO:23) (contains the promoter (base pairs 1-1704);5′UTR (base pairs 1705-1782); intron#1 (base pairs 1786-2190); and aportion of the fad2-1B coding region (base pairs 1783-1785 and2191-2463)) and its intron region (SEQ ID NO:24) were identified bycomparison to the soybean cDNA sequence using the Pustell comparisonprogram in Macvector. The intron sequence begins after the ATG startcodon and is 405 bases long.

[0075] 1B. Soybean Δ15 Desaturase (fad3)

[0076] The partial soybean fad 3 genomic sequence was PCR amplified fromsoybean DNA using primers 10632,5′-CUACUACUACUACTCGAGACAAAGCCTTTAGCCTATG-3′ (SEQ ID NO:11), and 10633:5′-CAUCAUCAUCAUGGATCCCATGTC TCTCTATGCAAG-3′ (SEQ ID NO:12). The ExpandLong Template PCR system (Boehringer Mannheim) was used according to themanufacturers directions. The resulting PCR products were cloned intothe vector pCR 2.1 (Invitrogen) and sequenced. The soy fad 3 partialgenomic clone sequence and the intron regions were confirmed bycomparisons to the soybean fad 3 CDNA sequence using the Pustell programin Macvector. From the identified partial genomic soybean fad3 sequence(SEQ ID NO:3), seven introns were identified (SEQ ID NO:4 (intron #1),SEQ ID NO:5 (intron #2), SEQ ID NO:6 (intron #3A), SEQ ID NO:7 (intron#4), SEQ ID NO:8 (intron #5), SEQ ID NO:25 (intron #3B) and SEQ ID NO:26(intron #3C)). Intron #1 is 192 base pairs long and is located betweenpositions 294 and 485, intron #2 is 348 base pairs long and is locatedbetween positions 576 and 923, intron #3A is 142 base pairs long and islocated between positions 991 and 1132, intron #3B is 98 base pairs longand is located between positions 1225 and 1322, intron #3C is 115 basepairs long and is located between positions 1509 and 1623, intron #4 is1231 base pairs long and is located between positions 1705 and 2935, andintron #5 is 626 base pairs long and is located between positions 3074and 3699.

Example 2 Expression Constructs

[0077] The soybean fad2-1A intron sequence was amplified via PCR usingthe fad2-1A partial genomic clone (SEQ ID NO:27) as a template andprimers 12701 (5′-ACGAATTCCTCGAGGTAAA TTAAATTGTGCCTGC-3′ (SEQ ID NO:13))and 12702 (5′-GCGAGATCTATCG ATCTGTGTCAAAGTATAAAC-3′ (SEQ ID NO:14)). Theresulting amplification products were cloned into the vector pCR 2.1(Invitrogen) and sequenced. The soyfad2-1A intron was then cloned intothe expression cassette, pCGN3892, in sense and antisense orientations.The vector pCGN3892 contains the soybean 7S promoter and a pea RBCS 3′.Both gene fusions were then separately ligated into pCGN9372, a vectorthat contains the CP4 gene regulated by the FMV promoter. The resultingexpression constructs (PCGN5469 sense and pCGN5471 antisense) were usedfor transformation of soybean using biolistic methods described below.

[0078] The soybean fad2-1B intron sequence was amplified via PCR usingthe fad2-1B partial genomic clone (SEQ ID NO:23) as a template andprimers 13883 (5′-GCGATCGATGTATGATGCTAAATTAAATTGTGCCTG-3′ (SEQ IDNO:30)) and 13876 (5′-GCGGAATTCCTGTGTCAAAGTATAAAGAAG-3′ (SEQ ID NO:31)).The resulting amplification products were cloned into the vector pCR 2.1(Invitrogen) and sequenced. The soyfad2-1B intron was fused to the 3′end of the soy fad 2-1A intron in plasmids pCGN5468 (contains thesoybean 7S promoter fused to the soy fad2-1A intron (sense) and a peaRBCS 3′) or pCGN5470 (contains the soybean 7S promoter fused to the soyfad2-1A intron (antisense) and a pea RBCS 3′) in sense or antisenseorientation respectively. The resulting intron combo fusions were thenligated separately into pCGN9372, a vector that contains the CP4 generegulated by the FMV promoter. The resulting expression constructs(pCGN5485, fad2-1A&B intron sense and pCGN5486, fad2-1A&B intronantisense) were used for transformation of soybean using biolisticmethods described below.

[0079] Four of the seven introns identified from the soybean fad 3genomic clone were PCR amplified using the soy fad 3 partial genomicclone as template and primers as follows: Intron #1, primers 12568:GATCGATGCCCGGGGTAATAATTTTTGTGT (SEQ ID NO:15) and 12569:CACGCCTCGAGTGTTCAATTCAATCAATG (SEQ ID NO:16); Intron #2, primers 12514:5′-CACTCGAGTTAGTTCATACTGGCT (SEQ ID NO:17) and 12515:5′-CGCATCGATTGCAAAATCCATCAAA (SEQ ID NO:18); Intron #4, primers 10926:5′-CUACUACUACUACTCGAGCGTAAATAGTGGGTGAACAC (SEQ ID NO:19) and 10927:5′-CAUCAUCAUCAUCTCGAGGAATTCGTCCATTTTAGTACACC (SEQ ID NO:20); Intron #5,primers 10928: 5′-CUACUACUACUACTCGAGGCGCGT ACATTTTATTGCTTA (SEQ IDNO:21) and 10929: 5′-CAUCAUCAUCAUCT CGAGGAATTCTGCAGTGAATCCAAATG (SEQ IDNO:22). The resulting PCR products for each intron were cloned into thevector pCR 2.1 (Invitrogen) and sequenced. Introns #1, #2, #4 and #5were all ligated separately into the, pCGN3892, in sense or antisenseorientations. pCGN3892 contains the soybean 7S promoter and a pea RBCS3′. These fusions were ligated into pCGN9372, a vector that contains theCP4 gene regulated by the FMV promoter for transformation into soybean.The resulting expression constructs (pCGN5455, fad3 intron#4 intronsense; pCGN5459, fad3 intron#4 intron antisense; pCGN5456, fad3 intron#5intron sense; pCGN5460, fad3 intron#5 intron antisense; pCGN5466, fad3intron#2 intron antisense; pCGN5473, fad3 intron#1 intron antisense;)were used for transformation of soybean using biolistic methodsdescribed below.

[0080] The soy fad3 Intron #3C and #4 were also PCR amplified from asecond fad3 gene family member, herein referred to as fad3-1B. The soyfad3-1B introns #3C and #4 were PCR amplified from soybean DNA using thefollowing primers, 5′ CATGCTTTCTGTGCTTCTC 3′ (SEQ ID NO:32) and, 5′GTTGATCCAACCATAGTCG 3′ (SEQ ID NO:33). The PCR products were cloned intothe vector pCR 2.1 (Invitrogen) and sequenced. The sequences for the soyfad3-1B introns #3C and #4 are provided in SEQ ID NOs:28 and 29.

Example 3 Plant Transformation and Analysis

[0081] Linear DNA fragments containing the expression constructs forsense and antisense expression of the Δ12 and Δ15 desaturase intronswere stably introduced into soybean (Asgrow variety A4922) by the methodof McCabe, et.al. (1988) Bio/Technology 6:923-926. Transformed soybeanplants were identified by selection on media containing glyphosate.

[0082] Fatty acid compositions were analyzed from seed of soybean linestransformed with the intron expression constructs using gaschromatography. T2 pooled seed and T2 single seed oil compositionsdemonstrate that the mono and polyunsaturated fatty acid compositionswere altered in the oil of seeds from transgenic soybean lines ascompared to that of the seed from non-transformed soybean. Table Iprovides a summary of results which were obtained using the describedconstructs. These data clearly show that sense and antisense expressionof the non-coding regions of the desaturase gene results in themodification of the fatty acid compositions. The data also shows thatintrons can be used to obtain a variety of lines with varying fatty acidcompositions. Selections can be made from such lines depending on thedesired relative fatty acid composition. In addition, since each of theintrons is able to modify the levels of each fatty acid to varyingextents, it is contemplated that combinations of introns can be useddepending on the desired compositions. TABLE I orien- tation event OleicLinoleic Linolenic Fad 2 wildtype 5469-5 null T2 pool 18.15% 55.59%7.97% (control) 10 seed average 13.89% 55.89% 9.067% 5469-27 null T2pool 19.15% 54.62% 9.32% A4922 15.75% 56.1% 8.75% 5471-13 null T2 pool17.02% 56.49% 9.08% 10 seed average 13.86% 56.14% 9.49% A4922 14.95%55.95% 9.07% full length sense 5462-133 T2 pool 84% 2.17% 1.55% cDNAbest 5462-133 T2 seed 84% 0.59% 1.76% (control) intron 1 sense 5469-6 T2pool 29.93% 46.53% 5469-8 T2 pool 36.5% 42.11% 5.98% best 5469-6 T2 seed44.41% 29.34% 6.68% best 5469-8 T2 seed 41.26% 33.16% 5.74% 5469-14 T2pool 61.06% 16.42% 7.75% 5469-20 T2 pool 48.89% 31.61% 4.89% 5469-22 T2pool 80% 2.97% 4.78% best 5469-14 T2 seed 62.21% 11.97% 8.81% 5485-3 T2pool 63.54% 14.09% 7.32% 5485-53 T2 pool 47.58% 27.64% 7.81% anti-5471-8 T2 pool 31.05% 43.62% 7.07% sense 5471-2 T2 pool 27.98% 48.88%6.83% 5471-26 T2 pool 32.66% 44.54% 6.76% best 5471-8 T2 seed 57.4%23.37% 5.73% best 5471-2 T2 seed 28.08% 46.14% 6.52% best 5471-26 T2seed 43.3% 34.15% 5.6% 5486-33 T2 pool 32.37% 43.66% 6.87% 5486-12 T2pool 27.32% 46.97% 6.4% 5486-40 T2 pool 26.79% 48.72% 6.55% Fad 3wildtype 5473-7 null T2 pool 15.65% 56.74% 9.55% (control) A4922 T2 pool19.84% 56.79% 7.48% full length sense 5464-50 T2 pool 18.06% 62.03%2.75% cDNA best 5464-50 T2 seed 17.08% 62.44% 1.72% (control) intron 1anti- 5473-8 T2 pool 33.47% 45.97% 5.54% sense 5473-1 T2 pool 33.34%42.67% 7.59% intron 2 anti- 5466-20 T2 pool 28.43% 48.83% 6.37% sense5466-16 T2 pool 27.61% 49.92% 5.96% intron 4 sense 5455-19 T2 pool40.35% 39.97% 4.61% 5455-10 T2 pool 35.14% 43.59% 5.53% 5455-57 T2 pool38.04% 42.44% 5.24% 5455-76 T2 pool 37.24% 42.42% 5.37% 5455-107 T2 pool36.44% 42.72% 5.62% best 5455-57 T2 seed 45.36% 35.55% 4.92% best5455-76 T2 seed 35.3% 43.54% 5.53% best 5455-107 T2 seed 45.56% 34.85%5.12% anti- 5459-2 T2 pool 34.5% 43.87% 5.59% sense 5459-6 T2 pool33.78% 44.12% 5.62% 5459-20 T2 pool 28.26% 49.48% 5.5% best 5459-2 T2seed 61.45% 23.45% 3.38% best 5459-6 T2 seed 53.51% 29.68% 3.53% best5459-20 T2 seed 30% 50.55% 4.15% intron 5 sense 5456-38 T2 pool 28.23%49.59% 6.74% 5456-62 T2 pool 28.94% 48.66% 6.25% best 5456-62 T2 seed29.5% 43.69% 5.4% anti- 5460-9 T2 pool 29.78% 48.57% 5.54% sense 5460-21T2 pool 28.37% 49.79% 5.54% best 5460-21 T2 seed 35.18% 40.52% 5.33%

[0083] All publications and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains. All publications and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0084] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1 33 1 4497 DNA Glycine max 1 cttgcttggt aacaacgtcg tcaagttattattttgttct tttttttttt atcatatttc 60 ttattttgtt ccaagtatgt catattttgatccatcttga caagtagatt gtcatgtagg 120 aataggaata tcactttaaa ttttaaagcattgattagtc tgtaggcaat attgtcttct 180 tcttcctcct tattaatatt ttttattctgccttcaatca ccagttatgg gagatggatg 240 taatactaaa taccatagtt gttctgcttgaagtttagtt gtatagttgt tctgcttgaa 300 gtttagttgt gtgtaatgtt tcagcgttggcttcccctgt aactgctaca atggtactga 360 atatatattt tttgcattgt tcatttttttcttttactta atcttcattg ctttgaaatt 420 aataaaacaa aaagaaggac cgaatagtttgaagtttgaa ctattgccta ttcatgtaac 480 ttattcaccc aatcttatat agtttttctggtagagatca ttttaaattg aaggatataa 540 attaagagga aatacttgta tgtgatgtgtggcaatttgg aagatcatgc gtagagagtt 600 taatggcagg ttttgcaaat tgacctgtagtcataattac actgggccct ctcggagttt 660 tgtgcctttt tgttgtcgct gtgtttggttctgcatgtta gcctcacaca gatatttagt 720 agttgttgtt ctgcatataa gcctcacacgtatactaaac gagtgaacct caaaatcatg 780 gccttacacc tattgagtga aattaatgaacagtgcatgt gagtatgtga ctgtgacaca 840 acccccggtt ttcatattgc aatgtgctactgtggtgatt aaccttgcta cactgtcgtc 900 cttgtttgtt tccttatgta tattgataccataaattatt actagtatat cattttatat 960 tgtccatacc attacgtgtt tatagtctctttatgacatg taattgaatt ttttaattat 1020 aaaaaataat aaaacttaat tacgtactataaagagatgc tcttgactag aattgtgatc 1080 tcctagtttc ctaaccatat actaatatttgcttgtattg atagcccctc cgttcccaag 1140 agtataaaac tgcatcgaat aatacaagccactaggcatg gtaaattaaa ttgtgcctgc 1200 acctcgggat atttcatgtg gggttcatcatatttgttga ggaaaagaaa ctcccgaaat 1260 tgaattatgc atttatatat cctttttcatttctagattt cctgaaggct taggtgtagg 1320 cacctagcta gtagctacaa tatcagcacttctctctatt gataaacaat tggctgtaat 1380 gccgcagtag aggacgatca caacatttcgtgctggttac tttttgtttt atggtcatga 1440 tttcactctc tctaatctct ccattcattttgtagttgtc attatcttta gatttttcac 1500 tacctggttt aaaattgagg gattgtagttctgttggtac atattacaca ttcagcaaaa 1560 caactgaaac tcaactgaac ttgtttatactttgacacag ggtctagcaa aggaaacaac 1620 aatgggaggt agaggtcgtg tggcaaagtggaagttcaag ggaagaagcc tctctcaagg 1680 gttccaaaca caaagccacc attcactgttggccaactca agaaagcaat tccaccacac 1740 tgctttcagc gctccctcct cacttcattctcctatgttg tttatgacct ttcatttgcc 1800 ttcattttct acattgccac cacctacttccacctccttc ctcaaccctt ttccctcatt 1860 gcatggccaa tctattgggt tctccaaggttgccttctca ctggtgtgtg ggtgattgct 1920 cacgagtgtg gtcaccatgc cttcagcaagtaccaatggg ttgatgatgt tgtgggtttg 1980 acccttcact caacactttt agtcccttatttctcatgga aaataagcca tcgccgccat 2040 cactccaaca caggttccct tgaccgtgatgaagtgtttg tcccaaaacc aaaatccaaa 2100 gttgcatggt tttccaagta cttaaacaaccctctaggaa gggctgtttc tcttctcgtc 2160 acactcacaa tagggtggcc tatgtatttagccttcaatg tctctggtag accctatgat 2220 agttttgcaa gccactacca cccttatgctcccatatatt ctaaccgtga gaggcttctg 2280 atctatgtct ctgatgttgc tttgttttctgtgacttact ctctctaccg tgttgcaacc 2340 ctgaaagggt tggtttggct gctatgtgtttatggggtgc ctttgctcat tgtgaacggt 2400 tttcttgtga ctatcacata tttgcagcacacacactttg ccttgcctca ttacgattca 2460 tcagaatggg actggctgaa gggagctttggcaactatgg acagagatta tgggattctg 2520 aacaaggtgt ttcatcacat aactgatactcatgtggctc accatctctt ctctacaatg 2580 ccacattacc atgcaatgga ggcaaccaatgcaatcaagc caatattggg tgagtactac 2640 caatttgatg acacaccatt ttacaaggcactgtggagag aagcgagaga gtgcctctat 2700 gtggagccag atgaaggaac atccgagaagggcgtgtatt ggtacaggaa caagtattga 2760 tggagcaacc aatgggccat agtgggagttatggaagttt tgtcatgtat tagtacataa 2820 ttagtagaat gttataaata agtggatttgccgcgtaatg actttgtgtg tattgtgaaa 2880 cagcttgttg cgatcatggt tataatgtaaaaataattct ggtattaatt acatgtggaa 2940 agtgttctgc ttatagcttt ctgcctaaaatgcacgctgc acgggacaat atcattggta 3000 atttttttaa aatctgaatt gaggctactcataatactat ccataggaca tcaaagacat 3060 gttgcattga ctttaagcag aggttcatctagaggattac tgcataggct tgaactacaa 3120 gtaatttaag ggacgagagc aactttagctctaccacgtc gttttacaag gttattaaaa 3180 tcaaattgat cttattaaaa ctgaaaatttgtaataaaat gctattgaaa aattaaaata 3240 tagcaaacac ctaaattgga ctgatttttagattcaaatt taataattaa tctaaattaa 3300 acttaaattt tataatatat gtcttgtaatatatcaagtt ttttttttta ttattgagtt 3360 tggaaacata taataaggaa cattagttaatattgataat ccactaagat cgacttagta 3420 ttacagtatt tggatgattt gtatgagatattcaaacttc actcttatca taatagagac 3480 aaaagttaat actgatggtg gagaaaaaaaaatgttattg ggagcatatg gtaagataag 3540 acggataaaa atatgctgca gcctggagagctaatgtatt ttttggtgaa gttttcaagt 3600 gacaactatt catgatgaga acacaataatattttctact tacctatccc acataaaata 3660 ctgattttaa taatgatgat aaataatgattaaaatattt gattctttgt taagagaaat 3720 aaggaaaaca taaatattct catggaaaaatcagcttgta ggagtagaaa ctttctgatt 3780 ataattttaa tcaagtttaa ttcattcttttaattttatt attagtacaa aatcattctc 3840 ttgaatttag agatgtatgt tgtagcttaatagtaatttt ttatttttat aataaaattc 3900 aagcagtcaa atttcatcca aataatcgtgttcgtgggtg taagtcagtt attccttctt 3960 atcttaatat acacgcaaag gaaaaaataaaaataaaatt cgaggaagcg cagcagcagc 4020 tgataccacg ttggttgacg aaactgataaaaagcgctgt cattgtgtct ttgtttgatc 4080 atcttcacaa tcacatctcc agaacacaaagaagagtgac ccttcttctt gttattccac 4140 ttgcgttagg tttctacttt cttctctctctctctctctc tcttcattcc tcatttttcc 4200 ctcaaacaat caatcaattt tcattcagattcgtaaattt ctcgattaga tcacggggtt 4260 aggtctccca ctttatcttt tcccaagcctttctctttcc ccctttccct gtctgcccca 4320 taaaattcag gatcggaaac gaactgggttcttgaatttc actctagatt ttgacaaatt 4380 cgaagtgtgc atgcactgat gcgacccactcccccttttt tgcattaaac aattatgaat 4440 tgaggttttt cttgcgatca tcattgcttgaattgaatca tattaggttt agattct 4497 2 420 DNA Glycine max 2 gtaaattaaattgtgcctgc acctcgggat atttcatgtg gggttcatca tatttgttga 60 ggaaaagaaactcccgaaat tgaattatgc atttatatat cctttttcat ttctagattt 120 cctgaaggcttaggtgtagg cacctagcta gtagctacaa tatcagcact tctctctatt 180 gataaacaattggctgtaat gccgcagtag aggacgatca caacatttcg tgctggttac 240 tttttgttttatggtcatga tttcactctc tctaatctct ccattcattt tgtagttgtc 300 attatctttagatttttcac tacctggttt aaaattgagg gattgtagtt ctgttggtac 360 atattacacattcagcaaaa caactgaaac tcaactgaac ttgtttatac tttgacacag 420 3 4010 DNAGlycine max 3 acaaagcctt tagcctatgc tgccaataat ggataccaac aaaagggttcttcttttgat 60 tttgatccta gcgctcctcc accgtttaag attgcagaaa tcagagcttcaataccaaaa 120 cattgctggg tcaagaatcc atggagatcc ctcagttatg ttctcagggatgtgcttgta 180 attgctgcat tggtggctgc agcaattcac ttcgacaact ggcttctctggctaatctat 240 tgccccattc aaggcacaat gttctgggct ctctttgttc ttggacatgattggtaataa 300 tttttgtgtt tcttactctt tttttttttt ttttgtttat gatatgaatctcacacattg 360 ttctgttatg tcatttcttc ttcatttggc tttagacaac ttaaatttgagatctttatt 420 atgtttttgc ttatatggta aagtgattct tcattatttc attcttcattgattgaattg 480 aacagtggcc atggaagctt ttcagatagc cctttgctga atagcctggtgggacacatc 540 ttgcattcct caattcttgt gccataccat ggatggttag ttcatactggcttttttgtt 600 tgttcatttg tcattgaaaa aaaatctttt gttgattcaa ttatttttatagtgtgtttg 660 gaagcccgtt tgagaaaata agaaatcgca tctggaatgt gaaagttataactatttagc 720 ttcatctgtc gttgcaagtt cttttattgg ttaaattttt atagcgtgctaggaaaccca 780 ttcgagaaaa taagaaatca catctggaat gtgaaagtta taactgttagcttctgagta 840 aacgtggaaa aaccacattt tggatttgga accaaatttt atttgataaatgacaaccaa 900 attgattttg atggattttg caggagaatt agccacagaa ctcaccatgaaaaccatgga 960 cacattgaga aggatgagtc atgggttcca gtatgtgatt aattgcttctcctatagttg 1020 ttcttgattc aattacattt tatttatttg gtaggtccaa gaaaaaagggaatctttatg 1080 cttcctgagg ctgttcttga acatggctct tttttatgtg tcattatcttagttaacaga 1140 gaagatttac aagaatctag acagcatgac aagactcatt agattcactgtgccatttcc 1200 atgtttgtgt atccaattta tttggtgagt gattttttga cttggaagacaacaacacat 1260 tattattata atatggttca aaacaatgac tttttcttta tgatgtgaactccatttttt 1320 agttttcaag aagccccgga aaggaaggct ctcacttcaa tccctacagcaatctgtttc 1380 cacccagtga gagaaaagga atagcaatat caacactgtg ttgggctaccatgttttctc 1440 tgcttatcta tctctcattc attaactagt ccacttctag tgctcaagctctatggaatt 1500 ccatattggg taactaaatt actcctacat tgttactttt tcctccttttttttattatt 1560 tcaattctcc aattggaaat ttgaaatagt taccataatt atgtaattgtttgatcatgt 1620 gcagatgttt gttatgtggc tggactttgt cacatacttg catcaccatggtcaccacca 1680 gaaactgcct tggtaccgcg gcaaggtaac aaaaataaat agaaaatagtgggtgaacac 1740 ttaaatgcga gatagtaata cctaaaaaaa gaaaaaaata taggtataataaataatata 1800 actttcaaaa taaaaagaaa tcatagagtc tagcgtagtg tttggagtgaaatgatgttc 1860 acctaccatt actcaaagat tttgttgtgt cccttagttc attcttattattttacatat 1920 cttacttgaa aagacttttt aattattcat tgagatctta aagtgactgttaaattaaaa 1980 taaaaaacaa gtttgttaaa acttcaaata aataagagtg aagggagtgtcatttgtctt 2040 ctttctttta ttgcgttatt aatcacgttt ctcttctctt ttttttttttcttctctgct 2100 ttccacccat tatcaagttc atgtgaagca gtggcggatc tatgtaaatgagtggggggc 2160 aattgcaccc acaagatttt attttttatt tgtacaggaa taataaaataaaactttgcc 2220 cccataaaaa ataaatattt tttcttaaaa taatgcaaaa taaatataagaaataaaaag 2280 agaataaatt attattaatt ttattatttt gtacttttta tttagtttttttagcggtta 2340 gatttttttt tcatgacatt atgtaatctt ttaaaagcat gtaatatttttattttgtga 2400 aaataaatat aaatgatcat attagtctca gaatgtataa actaataataattttatcac 2460 taaaagaaat tctaatttag tccataaata agtaaaacaa gtgacaattatattttatat 2520 ttacttaatg tgaaataata cttgaacatt ataataaaac ttaatgacaggagatattac 2580 atagtgccat aaagatattt taaaaaataa aatcattaat acactgtactactatataat 2640 attcgatata tatttttaac atgattctca atagaaaaat tgtattgattatattttatt 2700 agacatgaat ttacaagccc cgtttttcat ttatagctct tacctgtgatctattgtttt 2760 gcttcgctgt ttttgttggt caagggactt agatgtcaca atattaatactagaagtaaa 2820 tatttatgaa aacatgtacc ttacctcaac aaagaaagtg tggtaagtggcaacacacgt 2880 gttgcatttt tggcccagca ataacacgtg tttttgtggt gtactaaaatggacaggaat 2940 ggagttattt aagaggtggc ctcaccactg tggatcgtga ctatggttggatcaataaca 3000 ttcaccatga cattggcacc catgttatcc accatctttt cccccaaattcctcattatc 3060 acctcgttga agcggtacat tttattgctt attcacctaa aaacaatacaattagtacat 3120 ttgttttatc tcttggaagt tagtcatttt cagttgcatg attctaatgctctctccatt 3180 cttaaatcat gttttcacac ccacttcatt taaaataaga acgtgggtgttattttaatt 3240 tctattcact aacatgagaa attaacttat ttcaagtaat aattttaaaatatttttatg 3300 ctattatttt attacaaata attatgtata ttaagtttat tgattttataataattatat 3360 taaaattata tcgatattaa tttttgattc actgatagtg ttttatattgttagtactgt 3420 gcatttattt taaaattggc ataaataata tatgtaacca gctcactatactatactggg 3480 agcttggtgg tgaaaggggt tcccaaccct cctttctagg tgtacatgctttgatacttc 3540 tggtaccttc ttatatcaat ataaattata ttttgctgat aaaaaaacatggttaaccat 3600 taaattcttt ttttaaaaaa aaaactgtat ctaaactttg tattattaaaaagaagtctg 3660 agattaacaa taaactaaca ctcatttgga ttcactgcag acacaagcagcaaaaccagt 3720 tcttggagat tactaccgtg agccagaaag atctgcgcca ttaccatttcatctaataaa 3780 gtatttaatt cagagtatga gacaagacca cttcgtaagt gacactggagatgttgttta 3840 ttatcagact gattctctgc tcctccactc gcaacgagac tgagtttcaaactttttggg 3900 ttattattta ttgattctag ctactcaaat tacttttttt ttaatgttatgttttttgga 3960 gtttaacgtt ttctgaacaa cttgcaaatt acttgcatag agagacatgg4010 4 192 DNA Glycine max 4 gtaataattt ttgtgtttct tactcttttt tttttttttttgtttatgat atgaatctca 60 cacattgttc tgttatgtca tttcttcttc atttggctttagacaactta aatttgagat 120 ctttattatg tttttgctta tatggtaaag tgattcttcattatttcatt cttcattgat 180 tgaattgaac ag 192 5 348 DNA Glycine max 5gttagttcat actggctttt ttgtttgttc atttgtcatt gaaaaaaaat cttttgttga 60ttcaattatt tttatagtgt gtttggaagc ccgtttgaga aaataagaaa tcgcatctgg 120aatgtgaaag ttataactat ttagcttcat ctgtcgttgc aagttctttt attggttaaa 180tttttatagc gtgctaggaa acccattcga gaaaataaga aatcacatct ggaatgtgaa 240agttataact gttagcttct gagtaaacgt ggaaaaacca cattttggat ttggaaccaa 300attttatttg ataaatgaca accaaattga ttttgatgga ttttgcag 348 6 142 DNAGlycine max 6 gtatgtgatt aattgcttct cctatagttg ttcttgattc aattacattttatttatttg 60 gtaggtccaa gaaaaaaggg aatctttatg cttcctgagg ctgttcttgaacatggctct 120 tttttatgtg tcattatctt ag 142 7 1231 DNA Glycine max 7gtaacaaaaa taaatagaaa atagtgggtg aacacttaaa tgcgagatag taatacctaa 60aaaaagaaaa aaatataggt ataataaata atataacttt caaaataaaa agaaatcata 120gagtctagcg tagtgtttgg agtgaaatga tgttcaccta ccattactca aagattttgt 180tgtgtccctt agttcattct tattatttta catatcttac ttgaaaagac tttttaatta 240ttcattgaga tcttaaagtg actgttaaat taaaataaaa aacaagtttg ttaaaacttc 300aaataaataa gagtgaaggg agtgtcattt gtcttctttc ttttattgcg ttattaatca 360cgtttctctt ctcttttttt tttttcttct ctgctttcca cccattatca agttcatgtg 420aagcagtggc ggatctatgt aaatgagtgg ggggcaattg cacccacaag attttatttt 480ttatttgtac aggaataata aaataaaact ttgcccccat aaaaaataaa tattttttct 540taaaataatg caaaataaat ataagaaata aaaagagaat aaattattat taattttatt 600attttgtact ttttatttag tttttttagc ggttagattt ttttttcatg acattatgta 660atcttttaaa agcatgtaat atttttattt tgtgaaaata aatataaatg atcatattag 720tctcagaatg tataaactaa taataatttt atcactaaaa gaaattctaa tttagtccat 780aaataagtaa aacaagtgac aattatattt tatatttact taatgtgaaa taatacttga 840acattataat aaaacttaat gacaggagat attacatagt gccataaaga tattttaaaa 900aataaaatca ttaatacact gtactactat ataatattcg atatatattt ttaacatgat 960tctcaataga aaaattgtat tgattatatt ttattagaca tgaatttaca agccccgttt 1020ttcatttata gctcttacct gtgatctatt gttttgcttc gctgtttttg ttggtcaagg 1080gacttagatg tcacaatatt aatactagaa gtaaatattt atgaaaacat gtaccttacc 1140tcaacaaaga aagtgtggta agtggcaaca cacgtgttgc atttttggcc cagcaataac 1200acgtgttttt gtggtgtact aaaatggaca g 1231 8 626 DNA Glycine max 8gtacatttta ttgcttattc acctaaaaac aatacaatta gtacatttgt tttatctctt 60ggaagttagt cattttcagt tgcatgattc taatgctctc tccattctta aatcatgttt 120tcacacccac ttcatttaaa ataagaacgt gggtgttatt ttaatttcta ttcactaaca 180tgagaaatta acttatttca agtaataatt ttaaaatatt tttatgctat tattttatta 240caaataatta tgtatattaa gtttattgat tttataataa ttatattaaa attatatcga 300tattaatttt tgattcactg atagtgtttt atattgttag tactgtgcat ttattttaaa 360attggcataa ataatatatg taaccagctc actatactat actgggagct tggtggtgaa 420aggggttccc aaccctcctt tctaggtgta catgctttga tacttctggt accttcttat 480atcaatataa attatatttt gctgataaaa aaacatggtt aaccattaaa ttcttttttt 540aaaaaaaaaa ctgtatctaa actttgtatt attaaaaaga agtctgagat taacaataaa 600ctaacactca tttggattca ctgcag 626 9 18 DNA Artificial Sequence SyntheticOligonucleotide 9 atacaagcca ctaggcat 18 10 26 DNA Artificial SequenceSynthetic Oligonucleotide 10 gattggccat gcaatgaggg aaaagg 26 11 37 DNAArtificial Sequence Synthetic Oligonucleotide 11 cuacuacuac uactcgagacaaagccttta gcctatg 37 12 36 DNA Artificial Sequence SyntheticOligonucleotide 12 caucaucauc auggatccca tgtctctcta tgcaag 36 13 34 DNAArtificial Sequence Synthetic Oligonucleotide 13 acgaattcct cgaggtaaattaaattgtgc ctgc 34 14 33 DNA Artificial Sequence SyntheticOligonucleotide 14 gcgagatcta tcgatctgtg tcaaagtata aac 33 15 30 DNAArtificial Sequence Synthetic Oligonucleotide 15 gatcgatgcc cggggtaataatttttgtgt 30 16 29 DNA Artificial Sequence Synthetic Oligonucleotide 16cacgcctcga gtgttcaatt caatcaatg 29 17 24 DNA Artificial SequenceSynthetic Oligonucleotide 17 cactcgagtt agttcatact ggct 24 18 25 DNAArtificial Sequence Synthetic Oligonucleotide 18 cgcatcgatt gcaaaatccatcaaa 25 19 38 DNA Artificial Sequence Synthetic Oligonucleotide 19cuacuacuac uactcgagcg taaatagtgg gtgaacac 38 20 41 DNA ArtificialSequence Synthetic Oligonucleotide 20 caucaucauc auctcgagga attcgtccattttagtacac c 41 21 39 DNA Artificial Sequence Synthetic Oligonucleotide21 cuacuacuac uactcgaggc gcgtacattt tattgctta 39 22 41 DNA ArtificialSequence Synthetic Oligonucleotide 22 caucaucauc auctcgagga attctgcagtgaatccaaat g 41 23 1734 DNA Glycine max 23 actatagggc acgcgtggtcgacggcccgg gctggtcctc ggtgtgactc agccccaagt 60 gacgccaacc aaacgcgtcctaactaaggt gtagaagaaa cagatagtat ataagtatac 120 catataagag gagagtgagtggagaagcac ttctcctttt tttttctctg ttgaaattga 180 aagtgttttc cgggaaataaataaaataaa ttaaaatctt acacactcta ggtaggtact 240 tctaatttaa tccacactttgactctatat atgttttaaa aataattata atgcgtactt 300 acttcctcat tatactaaatttaacatcga tgattttatt ttctgtttct cttctttcca 360 cctacataca tcccaaaatttagggtgcaa ttttaagttt attaacacat gtttttagct 420 gcatgctgcc tttgtgtgtgctcaccaaat tgcattcttc tctttatatg ttgtatttga 480 attttcacac catatgtaaacaagattacg tacgtgtcca tgatcaaata caaatgctgt 540 cttatactgg caatttgataaacagccgtc cattttttct ttttctcttt aactatatat 600 gctctagaat ctctgaagattcctctgcca tcgaatttct ttcttggtaa caacgtcgtc 660 gttatgttat tattttattctatttttatt ttatcatata tatttcttat tttgttcgaa 720 gtatgtcata ttttgatcgtgacaattaga ttgtcatgta ggagtaggaa tatcacttta 780 aaacattgat tagtctgtaggcaatattgt cttctttttc ctcctttatt aatatatttt 840 gtcgaagttt taccacaaggttgattcgct ttttttgtcc ctttctcttg ttctttttac 900 ctcaggtatt ttagtctttcatggattata agatcactga gaagtgtatg catgtaatac 960 taagcaccat agctgttctgcttgaattta tttgtgtgta aattgtaatg tttcagcgtt 1020 ggctttccct gtagctgctacaatggtact gtatatctat tttttgcatt gttttcattt 1080 tttcttttac ttaatcttcattgctttgaa attaataaaa caatataata tagtttgaac 1140 tttgaactat tgcctattcatgtaattaac ttattcactg actcttattg tttttctggt 1200 agaattcatt ttaaattgaaggataaatta agaggcaata cttgtaaatt gacctgtcat 1260 aattacacag gaccctgttttgtgcctttt tgtctctgtc tttggttttg catgttagcc 1320 tcacacagat atttagtagttgttctgcat acaagcctca cacgtatact aaaccagtgg 1380 acctcaaagt catggccttacacctattgc atgcgagtct gtgacacaac ccctggtttc 1440 catattgcaa tgtgctacgccgtcgtcctt gtttgtttcc atatgtatat tgataccatc 1500 aaattattat atcatttatatggtctggac cattacgtgt actctttatg acatgtaatt 1560 gagtttttta attaaaaaaatcaatgaaat ttaactacgt agcatcatat agagataatt 1620 gactagaaat ttgatgacttattctttcct aatcatattt tcttgtattg atagccccgc 1680 tgtccctttt aaactcccgagagagtataa aactgcatcg aatattacaa gatg 1734 24 405 DNA Glycine max 24gtatgatgct aaattaaatt gtgcctgcac cccaggatat ttcatgtggg attcatcatt 60tattgaggaa aactctccaa attgaatcgt gcatttatat tttttttcca tttctagatt 120tcttgaaggc ttatggtata ggcacctaca attatcagca cttctctcta ttgataaaca 180attggctgta ataccacagt agagaacgat cacaacattt tgtgctggtt accttttgtt 240ttatggtcat gatttcactc tctctaatct gtcacttccc tccattcatt ttgtacttct 300catatttttc acttcctggt tgaaaattgt agttctcttg gtacatacta gtattagaca 360ttcagcaaca acaactgaac tgaacttctt tatactttga cacag 405 25 98 DNA Glycinemax 25 gtgagtgatt ttttgacttg gaagacaaca acacattatt attataatat ggttcaaaac60 aatgactttt tctttatgat gtgaactcca ttttttag 98 26 115 DNA Glycine max26 gtaactaaat tactcctaca ttgttacttt ttcctccttt tttttattat ttcaattctc 60caattggaaa tttgaaatag ttaccataat tatgtaattg tttgatcatg tgcag 115 27 778DNA Glycine max misc_feature (1)...(778) n = A,T,C or G 27 atacaagccactaggcatgg taaattaaat tgtgcctgca cctcgggata tttcatgtgg 60 ggttcatcatatttgttgag gaaaagaaac tcccgaaatt gaattatgca tttatatatc 120 ctttttcatttctagatttc ctgaaggctt aggtgtaggc acctagctag tagctacaat 180 atcagcacttctctctattg ataaacaatt ggctgtaatg ccgcagtaga ggacgatcac 240 aacatttcgtgctggttact ttttgtttta tggtcatgat ttcactctct ctaatctctc 300 cattcattttgtagttgtca ttatctttag atttttcact acctggttta aaattgaggg 360 attgtagttctgttggtaca tattacacat tcagcaaaac aactgaaact caactgaact 420 tgtttatactttgacacagg gtctagcaaa ggaaacaaca atgggaggta gaggtcgtgt 480 ggccaaagtggaagttcaag ggaagaagcc tctctcaagg gttccaaaca caaagccacc 540 attcactgttggccaactca agaaagcaat tccaccacac tgctttcagc gctccctcct 600 cacttcattctcctatgttg tttatgacct ttcatttgcc ttcattttct acattgccac 660 cacctacttccacctccttc ctcaaccctt ttccctcatt gcatggccaa tcaagccgaa 720 ttctgcagatatccatcaca tggcggcggn tggngnaggn ntntanaggg cccaattc 778 28 148 DNAGlycine max 28 gtaatctcac tctcacactt tctttataca tcgcacacca gtgtgggttatttgcaacct 60 acaccgaagt aatgccctat aattaatggg gttaacacat gtccaagtccaatattttgt 120 tcacttattt gaacttgaac atgtgtag 148 29 361 DNA Glycine max29 gtatcccatt taacacaatt tgtttcatta acattttaag agaatttttt tttcaaaata 60gttttcgaaa ttaagcaaat accaagcaaa ttgttagatc tacgcttgta cttgttttaa 120agtcaaattc atgaccaaat tgtcctcaca agtccaaacc gtccactatt ttattttcac 180ctactttata gcccaatttg tcatttggtt acttcagaaa agagaacccc atttgtagta 240aatatattat ttatgaatta tggtagtttc aacataaaac atatttatgt gcagttttgc 300catccttcaa aagaagatag aaacttactc catgttactc tgtctatatg taatttcaca 360 g361 30 36 DNA Glycine max 30 gcgatcgatg tatgatgcta aattaaattg tgcctg 3631 30 DNA Glycine max 31 gcggaattcc tgtgtcaaag tataaagaag 30 32 19 DNAGlycine max 32 catgctttct gtgcttctc 19 33 19 DNA Glycine max 33gttgatccaa ccatagtcg 19

What is claimed is:
 1. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:1; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:1 over the entire length of SEQ ID NO:1; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:1 over the entire length of SEQ ID NO:1; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:1 over the entire length of SEQ ID NO:1; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:1 over the entire length of SEQ ID NO:1; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:1 or a fragment thereof; and g) a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 2. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:2; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:2 or a fragment thereof; and g) a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 3. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:3; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:3 over the entire length of SEQ ID NO:3; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:3 over the entire length of SEQ ID NO:3; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:3 over the entire length of SEQ ID NO:3; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:3 over the entire length of SEQ ID NO:3; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:3 or a fragment thereof; and g) a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 4. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:4; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:4 over the entire length of SEQ ID NO:4; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:4 over the entire length of SEQ ID NO:4; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:4 over the entire length of SEQ ID NO:4; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:4 over the entire length of SEQ ID NO:4; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:4 or a fragment thereof; and g) a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 5. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:5; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:5 over the entire length of SEQ ID NO:5; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:5 over the entire length of SEQ ID NO:5; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:5 over the entire length of SEQ ID NO:5; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:5 over the entire length of SEQ ID NO:5; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:5 or a fragment thereof; and g) a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 6. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:6; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:6 over the entire length of SEQ ID NO:6; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:6 over the entire length of SEQ ID NO:6; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:6 over the entire length of SEQ ID NO:6; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:6 over the entire length of SEQ ID NO:6; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:6 or a fragment thereof; and g) a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 7. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:7; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:7 over the entire length of SEQ ID NO:7; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:7 over the entire length of SEQ ID NO:7; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:7 over the entire length of SEQ ID NO:7; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:7 over the entire length of SEQ ID NO:7; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:7 or a fragment thereof; and g) a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 8. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:8; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:8 over the entire length of SEQ ID NO:8; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:8 over the entire length of SEQ ID NO:8; d) a polynucleotide sequence having at least 90% identity to that of SEQ i) NO:8 over the entire length of SEQ ID NO:8; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:8 over the entire length of SEQ i) NO:8; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:8 or a fragment thereof; and g) a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 9. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:23; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:23 over the entire length of SEQ ID NO:23; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:23 over the entire length of SEQ ID NO:23; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:23 over the entire length of SEQ ID NO:23; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:23 over the entire length of SEQ ID NO:23; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:23 or a fragment thereof; and g) a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 10. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:24; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:24 over the entire length of SEQ ID NO:24; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:24 over the entire length of SEQ ID NO:24; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:24 over the entire length of SEQ ID NO:24; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:24 over the entire length of SEQ ID NO:24; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:24 or a fragment thereof; and g) a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 11. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:25; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:25 over the entire length of SEQ ID NO:25; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:25 over the entire length of SEQ ID NO:25; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:25 over the entire length of SEQ ID NO:25; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:25 over the entire length of SEQ ID NO:25; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:25 or a fragment thereof; and a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:26; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:26 over the entire length of SEQ ID NO:26; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:26 over the entire length of SEQ ID NO:26; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:26 over the entire length of SEQ ID NO:26; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:26 over the entire length of SEQ ID NO:26; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:26 or a fragment thereof; and a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 13. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:27; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:27 over the entire length of SEQ ID NO:27; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:27 over the entire length of SEQ ID NO:27; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:27 over the entire length of SEQ ID NO:27; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:27 over the entire length of SEQ ID NO:27; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:27 or a fragment thereof; and a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 14. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:28; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:28 over the entire length of SEQ ID NO:28; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:28 over the entire length of SEQ ID NO:28; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:28 over the entire length of SEQ ID NO:28; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:28 over the entire length of SEQ ID NO:28; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:28 or a fragment thereof; and a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 15. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising SEQ ID NO:29; b) a polynucleotide sequence having at least 70% identity to that of SEQ ID NO:29 over the entire length of SEQ ID NO:29; c) a polynucleotide sequence having at least 80% identity to that of SEQ ID NO:29 over the entire length of SEQ ID NO:29; d) a polynucleotide sequence having at least 90% identity to that of SEQ ID NO:29 over the entire length of SEQ ID NO:29; e) a polynucleotide sequence having at least 95% identity to that of SEQ ID NO:29 over the entire length of SEQ ID NO:29; f) an polynucleotide sequence that hybridizes, under stringent conditions, to SEQ ID NO:29 or a fragment thereof; and a polynucleotide sequence complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
 16. An intron obtained from a genomic polynucleotide sequence selected from the group consisting of: a) a genomic polynucleotide sequence having at least 70% identity to coding regions of SEQ ID NO:1 over the entire coding regions of SEQ ID NO:1; b) a genomic polynucleotide sequence having at least 80% identity to coding regions of SEQ ID NO:1 over the entire coding regions of SEQ ID NO:1; c) a genomic polynucleotide sequence having at least 90% identity to coding regions of SEQ ID NO:1 over the entire coding regions of SEQ ID NO:1; and d) a genomic polynucleotide sequence having at least 95% identity to coding regions of SEQ ID NO:1 over the entire coding regions of SEQ ID NO:1.
 17. An intron obtained from a genomic polynucleotide sequence selected from the group consisting of: a) a genomic polynucleotide sequence having at least 70% identity to coding regions of SEQ ID NO:3 over the entire coding regions of SEQ ID NO:3; b) a genomic polynucleotide sequence having at least 80% identity to coding regions of SEQ ID NO:3 over the entire coding regions of SEQ ID NO:3; c) a genomic polynucleotide sequence having at least 90% identity to coding regions of SEQ ID NO:3 over the entire coding regions of SEQ ID NO:3; and d) a genomic polynucleotide sequence having at least 95% identity to coding regions of SEQ ID NO:3 over the entire coding regions of SEQ ID NO:3.
 18. An intron obtained from a genomic polynucleotide sequence selected from the group consisting of: a) a genomic polynucleotide sequence having at least 70% identity to coding regions of SEQ ID NO:23 over the entire coding regions of SEQ ID NO:23; b) a genomic polynucleotide sequence having at least 80% identity to coding regions of SEQ ID NO:23 over the entire coding regions of SEQ ID NO:23; c) a genomic polynucleotide sequence having at least 90% identity to coding regions of SEQ ID NO:23 over the entire coding regions of SEQ ID NO:23; and d) a genomic polynucleotide sequence having at least 95% identity to coding regions of SEQ ID NO:23 over the entire coding regions of SEQ ID NO:23.
 19. An intron obtained from a genomic polynucleotide sequence selected from the group consisting of: a) a genomic polynucleotide sequence having at least 70% identity to coding regions of SEQ ID NO:27 over the entire coding regions of SEQ ID NO:27; b) a genomic polynucleotide sequence having at least 80% identity to coding regions of SEQ ID NO:27 over the entire coding regions of SEQ ID NO:27; c) a genomic polynucleotide sequence having at least 90% identity to coding regions of SEQ ID NO:27 over the entire coding regions of SEQ ID NO:27; and d) a genomic polynucleotide sequence having at least 95% identity to coding regions of SEQ ID NO:27 over the entire coding regions of SEQ ID NO:27.
 20. A recombinant DNA construct comprising at least one of the polynucleotide sequences of claims 1-19.
 21. The recombinant DNA construct according to claim 20, wherein said polynucleotide sequence is an intron sequence.
 22. The recombinant DNA construct according to claim 21, wherein said intron sequence is in an orientation selected from the group consisting of sense and antisense.
 23. A plant cell comprising the DNA construct of
 20. 24. A plant comprising the cell of claim
 24. 25. A method of modifying the fatty acid composition in a plant cell, said method comprising: transforming a plant cell with the construct of claim 21 and, growing said cell under conditions wherein transcription of said polynucleotide sequence is initiated, whereby said fatty acid composition of said cell is modified.
 26. The method according to claim 25, wherein said modification is selected from the group consisting of an increase in oleic acid, a decrease in linolenic acid and a decrease in linoleic acid.
 27. The method according to claim 25, wherein said polynucleotide sequence is an intron sequence.
 28. The method according to claim 27, wherein said intron sequence is in an orientation selected from the group consisting of sense and antisense.
 29. A plant cell produced by the method of claim
 25. 30. A method of inhibiting gene expression in a plant cell, comprising the steps of: transforming a plant cell with a DNA construct comprising a non-coding region of a gene to be inhibited positioned in a sense orientation; and growing said cell under conditions wherein transcription of said non-coding region is initiated, whereby said expression of said gene is inhibited.
 31. The method according to claim 30 wherein said non-coding region is selected from the group consisting of an intron, a promoter region, a 5′ untranslated region and portions of such sequences.
 32. The method according to claim 30 wherein said non-coding region is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:24, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:29.
 33. The method according to claim 30 wherein said gene encodes desaturase.
 34. A method of inhibiting desaturase expression in a plant cell, comprising the steps of: transforming a plant cell with a DNA construct comprising a non-coding region of a gene encoding desaturase positioned in an antisense orientation; and growing said cell under conditions wherein transcription of said non-coding region is initiated, whereby said expression of said desaturase is inhibited.
 35. The method according to claim 34 wherein said non-coding region is selected from the group consisting of an intron, a promoter region, a 5′ untranslated region and portions of such sequences.
 36. The method according to claim 34 wherein said non-coding region is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:24, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:29.
 37. A plant cell produced according to the method of claim
 34. 38. The plant cell according to claim 37 wherein said cell includes an oil composition comprising at least about 80-85% oleic acid, no more than about 1-2% linoleic acid and no more than about 1-3% linolenic acid.
 39. The plant cell according to claim 37 wherein said cell includes an oil composition comprising at least about 50-75% oleic acid, no more than about 10-30% linoleic acid and no more than about 1-3% linolenic acid.
 40. A method of modifying fatty acid composition in a plant cell, comprising the steps of: transforming a plant cell with a DNA construct comprising a non-coding region of a gene encoding desaturase positioned in a sense or an antisense orientation; and growing said cell under conditions wherein transcription of said non-coding region is initiated, whereby said expression of said desaturase is inhibited.
 41. The method according to claim 40 wherein said non-coding region is selected from the group consisting of an intron, a promoter region, a 5′ untranslated region and portions of such sequences.
 42. The method according to claim 41 wherein said non-coding region is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:24, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:29.
 43. A plant cell produced according to the method of claim
 40. 44. The plant cell according to claim 43 wherein said cell includes an oil composition comprising at least about 80-85% oleic acid, no more than about 1-2% linoleic acid and no more than about 1-3% linolenic acid.
 45. The plant cell according to claim 43 wherein said cell includes an oil composition comprising at least about 50-75% oleic acid, no more than about 10-30% linoleic acid and no more than about 1-3% linolenic acid.
 46. A method of inhibiting desaturase expression in a plant cell, comprising the steps of: transforming a plant cell with a DNA construct comprising a nucleic acid sequence capable of binding to or cleaving a non-coding region of a gene encoding desaturase; and growing said cell under conditions wherein transcription of said nucleic acid sequence is initiated, whereby said expression of said desaturase is inhibited.
 47. A plant cell produced according to the method of claim
 46. 48. A oil seed composition comprising: at least about 50-75% oleic acid, no more than about 10-30% linoleic acid and no more than about 1-3% linolenic acid. 